Primary Vascular Endothelial Cells Research Articles

Phosphorylation of Janus kinase 1 (JAK1) by AMP-activated protein kinase (AMPK) links energy sensing to anti-inflammatory signaling.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a pivotal regulator of metabolism at cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser515 and Ser518) within the Src homology 2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect that required the presence of Ser515 and Ser518 and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser515 and Ser518 abolished AMPK-mediated inhibition of JAK-STAT signaling stimulated by either the sIL-6Rα/IL-6 complex or the expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore, our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.

CTRP9 induces mitochondrial biogenesis and protects high glucose-induced endothelial oxidative damage via AdipoR1 -SIRT1- PGC-1α activation

Vascular lesions caused by endothelial dysfunction are the most common and serious complication of diabetes. The vasoactive potency of CTRP9 has been reported in our previous study via nitric oxide (NO) production. However, the effect of CTRP9 on vascular endothelial cells remains unknown. This study aimed to investigate the protection role of CTRP9 in the primary aortic vascular endothelial cells and HAECs under high-glucose condition. We found that the aortic vascular endothelial cells isolated from mice fed with a high fat diet generated more ROS production than normal cells, along with decreased mitochondrial biogenesis, which was also found in HAECs treated with high glucose. However, the treatment of CTPR9 significantly reduced ROS production and increased the activities of endogenous antioxidant enzymes, the expression of PGC-1α, NRF1, TFAM, ATP5A1 and SIRT1, and the activity of cytochrome c oxidase, indicating an induction of mitochondrial biogenesis. Furthermore, silencing the expression of SIRT1 in HAECs impeded the effect of CTRP9 on mitochondrial biogenesis, while silencing the expression of AdipoR1 in HAECs reversed the expression of SIRT1 and PGC-1α. Based on these findings, this study showed that CTRP9 might induce mitochondrial biogenesis and protect high glucose-induced endothelial oxidative damage via AdipoR1-SIRT1-PGC-1α signaling pathway.

Paeonol affects proliferation activity of rat vasular endothelial cells induced by lipopolysaccharide and co-cultured with smooth muscle cells via inhibiting pathway of PI3K/AKT-NF-κB signaling

This paper was aimed to observe the anti-atheroslerosis effect of paeonol (Pae) on the activation of PI3K/AKT-NF-κB and the proliferation activity of rat vasular endothelial cells induced by lipopolysaccharide (LPS) and co-cultured with smooth muscle cells. Primary rat vascular endothelial cells (VECs) and rat vascular smooth cells (VSMCs) were cultured by predigesting and adhering tissue blocks. The VEC-VSMC co-culture model was established by Transwell chamber. LPS (100 μg•L ⁻¹, 7 h) was used to induce VEC injury. MTT assay were used to determine the VEC proliferation activity. Western blot was used to detect PI3K/AKT and NF-κB's signaling pathways related protein expressions. The concentration of LPS-induced VECs injury was 100 μg•L ⁻¹, and the time was 7 h. After the intervention on the above cell model for 24 h, paeonol (15, 30, 60 μmol•L ⁻¹) could effectively inhibit LPS-induced VECs injury, block PI3K/AKT-NF-κB signal transduction pathway thereby significantly affecting the proliferation of LPS-induced VECs co-cultured with SMCs. The anti-atherosclerosis mechanism of paeonol may be related to the reducing the inhibitory effect of the signaling pathway associated proteins of VEC PI3K/AKT and NF-κB, thereby increasing the VEC livability under co-culture.

Decellularized matrices enriched with antibiotics: a promising engineering approach for tissue regeneration

Event Abstract Back to Event Decellularized matrices enriched with antibiotics: a promising engineering approach for tissue regeneration Francesca Boccafoschi1, Luca Fusaro1, Margherita Botta1, Francesca Torri1, Martina Ramella1, Francesco Copes1, Andrea Cochis1, Barbara Azzimonti1 and Mario Cannas1 1 University of Piemonte Orientale, Health Science Department, Italy Introduction: During modern age, transplantation techniques changed reconstructive surgery scenario, making remarkable improvements concerning the functionality of transplanted organs or tissues. Transplantations show also several pitfalls, such as limited tissue availability and multiple surgeries needed. Moreover, one of the main causes of prosthetic transplants failure is the in situ infection of the surgical site, caused by bacterial colonization that can occur during the operating procedures [1]. For these reasons, decellularization of native tissues has gained a significant attention especially for obtaining biological scaffolds that maintain the native tissues’ ECM structure, thus preserving their biomechanical properties. In order to optimize the post-surgical rehabilitation process, the biological substitutes can be enriched with several functional molecules and/or repopulated with different cell types. Materials and Methods: Our approach is based on antibiotic enrichment of decellularized bovine pericardium scaffolds Antibiotic (4% gentamicin) has been covalently bound on the substitute surface in order to inhibit the colonization of Staphylococcus aureus, a dangerous nosocomial bacterial strain, until complete healing, and thus favour the post-surgical tissue regeneration. Evaluation of decellularization and enrichment was performed with DAPI, XPS and ToF-SIMS MS analyses. Sensitivity tests (inhibition halo, XTT assay) towards 4% gentamicin of Staphylococcus aureus were performed. In order to verify cell sensitivity of tissues interacting with the scaffold, toxicity tests on primary porcine vascular endothelial and smooth muscle cells, were performed. The biomechanical and biocompatibility properties of decellularized and enriched matrices have been characterized, with tensile stress and Young modulus analyses, and MTT assay. Results and Discussion: The DAPI staining of the analyzed scaffold confirmed the absence of nuclei. Analytical chemistry tests confirmed the efficiency of enrichment method. A significant difference, in terms of inhibition halo formation, between gentamicin (4%) coated and grafted decellularized scaffolds, was observed: a large inhibition halo for the 4% gentamicin covalently enriched samples has been shown. XTT assay showed a decrease of bacterial viability for the 4% gentamicin covalently enriched samples towards other samples. Regarding the comparison between the mechanical properties of native and decellularized matrices, no significant differences were found in Young moduli. MTT assay showed similar cell viability values for gentamicin enriched samples and untreated controls, meaning that treated materials do not inhibit tissue regeneration. Conclusion: Our tests confirm the efficiency of the “decellularized tissue” method and its ability to maintain adequate mechanical properties. Thus the enriched biological substitutes could represent an innovative engineering approach for surgical tissue regeneration. We would like to thank the Assut S.p.A for financial support.

Shear stress mediates exocytosis of functional TRPV4 channels in endothelial cells.

Mechanosensitive ion channels are implicated in the biology of touch, pain, hearing and vascular reactivity; however, the identity of these ion channels and the molecular basis of their activation is poorly understood. We previously found that transient receptor potential vanilloid 4 (TRPV4) is a receptor operated ion channel that is sensitised and activated by mechanical stress. Here, we investigated the effects of mechanical stimulation on TRPV4 localisation and activation in native and recombinant TRPV4-expressing cells. We used a combination of total internal reflection fluorescence microscopy, cell surface biotinylation assay and Ca(2+) imaging with laser scanning confocal microscope to show that TRPV4 is expressed in primary vascular endothelial cells and that shear stress sensitises the response of TRPV4 to its agonist, GSK1016790A. The sensitisation was attributed to the recruitment of intracellular pools of TRPV4 to the plasma membrane, through the clathrin and dynamin-mediated exocytosis. The translocation was dependent on ILK/Akt signalling pathway, release of Ca(2+) from intracellular stores and we demonstrated that shear stress stimulated phosphorylation of TRPV4 at tyrosine Y110. Our findings implicate calcium-sensitive TRPV4 translocation in the regulation of endothelial responses to mechanical stimulation.

Cardiac and Vascular Synergic Protective Effect of Olea europea L. Leaves and Hibiscus sabdariffa L. Flower Extracts.

This study was aimed at investigating the cardiovascular effects of an Olea europea L. leaf extract (OEE), of a Hibiscus sabdariffa L. flower extract (HSE), and of their 13 : 2 w/w mixture in order to assess their cardiac and vascular activity. Both extracts were fully characterized in their bioactive compounds by HPLC-MS/MS analysis. The study was performed using primary vascular endothelial cells (HUVECs) to investigate the antioxidant and cytoprotective effect of the extracts and their mixture and isolated guinea-pig left and right atria and aorta to evaluate the inotropic and chronotropic activities and vasorelaxant properties. In cultured HUVECs, OEE and HSE reduced intracellular reactive oxygen species formation and improved cell viability, following oxidative stress in dose-dependent manner. OEE and HSE exerted negative inotropic and vasorelaxant effects without any chronotropic property. Interestingly, the mixture exerted higher cytoprotective effects and antioxidant activities. Moreover, the mixture exerted an inotropic effect similar to each single extract, while it revealed an intrinsic negative chronotropic activity different from the single extract; its relaxant activity was higher than that of each single extract. In conclusion OEE and HSE mixture has a good potential for pharmaceutical and nutraceutical application, thanks to the synergistic effects of the single phytochemicals.

ASSA14-03-31 CREG modulates Angiotensin II type 1 receptor to antagonise coronary oxidative stress and hypertension in salt-sensitive rats

Background This study examined whether CREG protected Sprague-Dawley (SD) rats against high salt-related hypertension and vascular endothelium oxidative stress, and explored the mechanisms and associated changes in components of the renin-angiotensin system and coronary artery injury. Methods In this study, SD rats received pure drinking water, or drinking water containing 1.5% NaCl for 16 weeks. Systolic blood pressure was measured before rats were divided into salt-sensitive and salt-resistant groups. After 16 weeks of treatment, NO, malondialdehyde (MDA), angiotensin II and IL-6 in plasma and angiotensin II in coronary arteries were detected, as was the expression of eNOS, AT1R, AT2R and CREG. Primary lung vascular endothelial cells were incubated in culture medium containing aldosterone (0.45 nmol/l). Salt-induced changes in eNOS and activation of NAD(P)H oxidase-gp91 were assessed. Supernatant ONOO- and NO, and the expression of AT1R, AT2R and CREG were measured. Losartan was used to block AT1R effects, and to investigate the biofunction of CREG on endothelial cells. Results The results showed that a long-term high salt intake significantly increased plasma IL-6 and MDA, angiotensin II in coronary arteries and AT1R expression, but decreased plasma NO, angiotensin II, AT2R and CREG expression in coronary arteries of salt-sensitive rats, which was prior to increase of blood pressure. Conversely, no obvious oxidative stress or vascular remodelling was observed in the salt-resistance group accompanying anti-hypertension. Mechanically, sodium concentrations significantly increased the expression of gp91 (a marker of oxidative stress) and AT1R, and reduced expression of eNOS and CREG in primary lung vascular endothelial cells from salt-sensitive rats. These effects were abrogated in cells with overexpression of CREG. Furthermore, Losartan blocking the biofunction of AT1R antagonised the NaCl-induced oxidative stress in CREG-silencing endothelial cells. Conclusions This is the first study to identify CREG-modulated AT1R activation as a novel target to block oxidative stress in blood vessels and prevent salt-related hypertension.

Symposium 3: Inflammation: a key driving force in disease progression S3-1 - Control of endothelial function and angiogenesis by PGC-1α relies on ROS control of vascular stability

Peroxisome proliferator activated receptor g co-activator 1alpha (PGC-1α) is a regulator of oxidative metabolism and reactive oxygen species (ROS) homeostasis that has been show to play a relevant role in angiogenesis. PGC-1α KO mice show reduced vascular density in the retinas and KO primary vascular endothelial cells (ECs) migrate faster than the wild type, an effect that can be rescued by antioxidants, suggesting that excessive ROS levels might be relevant in PGC-1 α role in angiogenesis. This study aims to investigate the role of ROS homeostasis on the regulation by PGC-1 α of angiogenesis. We found that endothelial cells (ECs) from mice deleted for PGC-1 α display attenuated adhesion to the extracellular matrix, together with slower spreading, reduced formation of cellular junctions, a disorganized cytoskeleton and random motility, and a enhanced tip phenotype. Aditionally, PGC-1 α -deleted ECs exhibit an altered response to vascular endothelial growth factor-A (VEGF-A). In vivo, deletion of PGC-1 α results in addition to reduced retinal vascular density, sparse pericyte coverage. Exposure of PGC-1 α deleted mice to hyperoxia during retinal vascular development exacerbates these vascular abnormalities and mice show extensive retinal hemorrhaging, with highly unstructured areas and very poor perfusion, compared with wild-type mice. Structural analysis demonstrates a reduction of endothelial VE-cadherin, suggesting defective inter-cellular junctions. Interestingly, this hyperoxia-induced phenotype is partially reversed by antioxidant administration, indicating that elevated production of mitochondrial reactive oxygen species (ROS) in the absence of PGC-1 α is functionally important. Finally, in vitro studies show that antioxidant treatment improves VEGF-A signaling, suggesting that toxic effect of ROS may be caused by the alteration of the VEGF-A signaling pathway. In summary, our findings indicate that PGC-1 α control of ROS homeostasis plays an important role in the control of de novo angiogenesis, and is required for vascular stability.

Bisphenol A disrupts transcription and decreases viability in aging vascular endothelial cells.

Bisphenol A (BPA) is a widely utilized endocrine disruptor capable of mimicking endogenous hormones, employed in the manufacture of numerous consumer products, thereby interfering with physiological cellular functions. Recent research has shown that BPA alters epigenetic cellular mechanisms in mammals and may be correlated to enhanced cellular senescence. Here, the effects of BPA at 10 ng/mL and 1 µg/mL, concentrations found in human samples, were analyzed on HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) transcriptional analysis of the Long Interspersed Element-1 (LINE-1) retroelement showed that BPA induces global transcription deregulation in both cell lines, although with more pronounced effects in HUVEC cells. Whereas there was an increase in global transcription in HT29 exclusively after 24 h of exposure, this chemical had prolonged effects on HUVEC. Immunoblotting revealed that this was not accompanied by alterations in the overall content of H3K9me2 and H3K4me3 epigenetic marks. Importantly, cell viability assays and transcriptional analysis indicated that prolonged BPA exposure affects aging processes in senescent HUVEC. To our knowledge this is the first report that BPA interferes with senescence in primary vascular endothelial cells, therefore, suggesting its association to the etiology of age-related human pathologies, such as atherosclerosis.

Regulation of Store‐Operated Ca2+ Entry in the Vascular Endothelium by the G Protein‐coupled Estrogen Receptor 1 (LB570)

The novel G protein‐coupled estrogen receptor 1 (GPER/GPR30) has been found to participate in numerous cardiovascular functions. Store‐operated Ca2+ entry (SOCE) is an essential mechanism that is required for many endothelial cell functions. We found that activation of GPER using the GPER specific agonist G1 causes a dose‐dependent inhibition of SOCE in primary vascular endothelial cells. Heterologous of GPER in HEK 293 cells is associated with substantial decreases in the total Ca2+ signal triggered by thapsigargin, and a 40% decrease in the rate of SOCE. Knockdown of GPER in endothelial cells using antisense directed against GPER increases SOCE by approximately 50% compared to cells transfected scrambled oligo. Interestingly, GPER specific antagonist G15 triggers total Ca2+ signals via SOCE by releasing the same internal Ca2+ stores as does thapsigargin. In primary endothelial cells, GPER forms a complex with the stromal interaction molecule 1 (Stim1), an essential molecular switcher of SOCE. In addition, Stim1‐ECFP colocalizes with GPER‐tetracysteine when heterologously expressed in HEK 293 cells. Heterologous expression of GPER‐DsRed2 fusion substantially decreases total Ca2+ signal stimulated by thapsigargin. Interestingly, SOCE measured in cells stably expressing fusions between EYFP and individual sub‐membrane domains (SMDs) 1 ‐ 4 of GPER does not differ from internal controls. These data indicate that GPER may be an important regulatory input of store‐operated Ca2+ entry via its interaction with Stim1. Since the EYFP‐SMD fusions are all cytoplasmically expressed, the data may also suggest that proper membrane docking (PM or ER) of full‐length GPER is required for it to exert its effect on SOCE.Grant Funding Source: National Institutes of Health

The G Protein‐Coupled Estrogen Receptor 1 Regulates Endothelial Ca2+ efflux via the plasma membrane Ca2+‐ATPase. (LB568)

The G protein‐coupled estrogen receptor 1 (GPER) has been demonstrated to have a vast array of cardiovascular effects. Ca2+ extrusion via the plasma membrane Ca2+‐ATPase (PMCA) plays important roles in Ca2+ homeostasis. We tested the hypothesis that GPER is involved in the regulation of calcium efflux in the vasculature. In primary vascular endothelial cells, the specific GPER agonist G‐1 dose‐dependently inhibits PMCA activity. Knockdown of GPER in endothelial cells using antisense directed against GPER is associated with a ~ 40% enhancement of PMCA activity. Heterologous expression of human GPER in fusion with the fluorescent protein DsRed2 in HEK 293 cells results in a 38% reduction of PMCA activity. Co‐IP experiments show that PMCA and GPER form a complex during Ca2+ signals triggered by the SERCA pump inhibitor thapsigargin in endothelial cells. In addition, the fusion proteins PMCA‐DsRed2 colocalizes with GPER‐ECFP heterologously expressed in HEK 293 cells. These data suggest that GPER regulates Ca2+ efflux in endothelial cells by interacting with PMCA.Grant Funding Source: National Institutes of Health

Assessing the effect of shear stress on Aquaporin 1 expression in vascular endothelial cells in vitro (696.9)

The human saphenous vein (HSV) is commonly used in coronary artery bypass grafts. The patency of the vein graft in an arterial environment is limited, thereby requiring a high percentage of autograft recipients to repeat the bypass surgery within 5 years. The main problem that ensues with HSV grafts is due to the development of intimal hyperplasia (IH) which compromises vessel function. The mechanistic reasons for the development of IH and limited HSV patency are not currently understood. However, it has been proposed that the change from venous to arterial shear stress may be a trigger. Aquaporin 1 (AQP1), a water channel protein, is expressed in the plasma membrane of vascular endothelial cells. It is hypothesized that enhanced AQP1 expression may be an early biomarker for the development of IH. The goal of this study was to assess the effect of shear stress on AQP1 expression in cultured endothelial cells. Primary vascular endothelial cells seeded on a gelatin‐coated Ibidi flow chamber grown in static conditions expressed low levels of AQP1 protein that localized around the nucleus. In contrast, AQP1 was present in vesicles throughout the cytoplasm in increasing abundance in cells subjected to low shear stress (6 dynes/cm²) vs. high shear stress (16 dynes/cm²)(p<0.01). These data show that shear stress alters AQP1 abundance and subcellular distribution in vascular endothelial cells in vitro, thus supporting a role for AQP1 as an environmental sensor in HSV grafts.Grant Funding Source: Supported by the University of Dayton Research Council

Effect of paeonol on adhesive function of rat vascular endothelial cells induced by lipopolysaccharide and co-cultured with smooth muscle cells

To observe the changes in the adhesive function of vascular endothelial cells (VEC) and rat monocytes induced by lipopolysaccharide (LPS) and co-cultured with smooth muscle cells (SMC) and the intervention effect of paeonol (Pae). Primary rat vascular endothelial cells (VECs) and rat vascular smooth muscle cells (VSMCs) were cultured by predigesting and adhering tissue blocks. The VEC-VSMC co-culture model was established by Transwell chamber. LPS was used to induce VEC injury. MTT assay and LDH assay were used to determine the VEC activity. ELISA assay was used to detect IL-1beta and TNF-alpha secreted by the VEC. The immunocytochemistry assay was carried out to detect the expression of ICAM-1. The Rose Bengal Staining was used to test adhesive function between VECs and monocytes. The concentration of LPS-induced VEC injury was 100 microg x L(-1), and the time was 7 h. after the intervention on the above cell model for 24 h, Paeonol (15, 30, 60 micromol x L(-1)) could effectively inhibit LPS-induced VEC injury and VEC injury, significantly enhance the survival rate of LPS-injured VECs, decrease IL-1beta and TNF-alpha secreted by the injured VEC, and reduce the expression of ICAM-1, so as to inhibit the adhesion of LPS-induced VECs and monocytes. Paeonol could inhibit IL-1beta and TNF-alpha expression to protect VECs from being injured by LPS, and reduce ICAM-1 expression to inhibit the adhesion between VECs and monocytes.

Effective siRNA delivery to inflamed primary vascular endothelial cells by anti-E-selectin and anti-VCAM-1 PEGylated SAINT-based lipoplexes

The endothelium represents an attractive therapeutic target due to its pivotal role in many diseases including chronic inflammation and cancer. Small interfering RNAs (siRNAs) specifically interfere with the expression of target genes and are considered an important new class of therapeutics. However, due to their size and charge, siRNAs do not spontaneously enter unperturbed endothelial cells (EC). To overcome this problem, we developed novel lipoplexes for siRNA delivery that are based on the cationic amphiphilic lipid SAINT-C18. Antibodies recognizing disease induced cell adhesion molecules were employed to create cell specificity resulting in so-called antibody-SAINTargs. To improve particle stability, antibody-SAINTargs were further optimized for EC-specific siRNA-mediated gene silencing by addition of polyethylene glycol (PEG). Although PEGylated antibody-SAINTargs maintained specificity, they lost their siRNA delivery capacity. Coupling of antibodies to the distal end of PEG (so-called antibody-SAINTPEGargs), resulted in anti-E-selectin- and anti-vascular cell adhesion molecule (VCAM)-1-SAINTPEGarg that preserved their antigen recognition and their capability to specifically deliver siRNA into inflammation-activated primary endothelial cells. The enhanced uptake of siRNA by antibody-SAINTPEGargs was followed by improved silencing of the target gene VE-cadherin, demonstrating that antibody-SAINTPEGargs were capable of functionally delivering siRNA into primary endothelial cells originating from different vascular beds. In conclusion, the newly developed, physicochemically stable, and EC-specific siRNA carrying antibody-SAINTPEGargs selectively down-regulate target genes in primary endothelial cells that are generally difficult to transfect.

Regulation of Store‐Operated Ca2+ Entry in the Vascular Endothelium by the G Protein‐Coupled Estrogen Receptor

The novel G protein‐coupled estrogen receptor (GPER/GPR30) has been found to participate in numerous cardiovascular functions. Store‐operated Ca2+ entry (SOCE) is an essential mechanism that is required for many endothelial cell functions. We found that activation of GPER using the GPER specific agonist G1 is associated with a dose‐dependent inhibition of SOCE in primary vascular endothelial cells. Interestingly, the GPER specific antagonist G15 increases SOCE in cells unstimulated by GPER intrinsic or exogenous ligand. Overexpression of GPER in HEK 293 cells is associated with a 40% decrease in the rate of SOCE, while knockdown of GPER in endothelial cells using shRNA or antisense oligomer directed against GPER increases SOCE by approximately 50%. In addition, coimmunoprecipitation revealed that GPER exists in endothelial cells as a glycosylated protein and forms a complex with the stromal interaction molecule 1 (Stim1). These data suggest that GPER may be an important regulatory input of store‐operated Ca2+ entry via its interaction with Stim1.

Identification of three distinct calmodulin‐binding domains in the G Protein‐coupled Estrogen Receptor

The novel G protein‐coupled estrogen receptor (GPER/GPR30) is being increasingly identified as an important GPCR in many organ system. Calmodulin (CaM) is a ubiquitous Ca2+ transducer that is required for the activities of numerous cellular proteins. Here we show that CaM coimmunoprecipitates with GPER in primary vascular endothelial cells upon stimulation with 17β‐estradiol or Ca2+‐elevating ionophore. To identify the CaM‐binding sites in GPER/GPR30, we have developed a series of novel FRET‐based biosensors whose responses allow identification and highly quantitative characterization of CaM‐binding properties of CaM‐binding domains in GPER/GPR30. Three separate CaM‐binding domains with distinct dissociation constants and Ca2+ sensitivities for complex formation with CaM were found. The functional implications of CaM‐GPER interactions are explored.

PCB 77 dechlorination products modulate pro-inflammatory events in vascular endothelial cells

Persistent organic pollutants such as polychlorinated biphenyls (PCBs) are associated with detrimental health outcomes including cardiovascular diseases. Remediation of these compounds is a critical component of environmental policy. Although remediation efforts aim to completely remove toxicants, little is known about the effects of potential remediation byproducts. We previously published that Fe/Pd nanoparticles effectively dechlorinate PCB 77 to biphenyl, thus eliminating PCB-induced endothelial dysfunction using primary vascular endothelial cells. Herein, we analyzed the toxic effects of PCB congener mixtures (representative mixtures of commercial PCBs based on previous dechlorination data) produced at multiple time points during the dechlorination of PCB 77 to biphenyl. Compared with pure PCB 77, exposing endothelial cells to lower chlorinated PCB byproducts led to improved cellular viability, decreased superoxide production, and decreased nuclear factor kappa B activation based on duration of remediation. Presence of the parent compound, PCB 77, led to significant increases in mRNA and protein inflammatory marker expression. These data implicate that PCB dechlorination reduces biological toxicity to vascular endothelial cells.

Titanium dioxide nanoparticles increase inflammatory responses in vascular endothelial cells

Atherosclerosis is a chronic inflammatory disease that remains the leading cause of death in the United States. Numerous risk factors for endothelial cell inflammation and the development of atherosclerosis have been identified, including inhalation of ultrafine particles. Recently, engineered nanoparticles (NPs) such as titanium (TiO2) NPs have attracted much attention due to their wide range of applications. However, there are also great concerns surrounding potential adverse health effects in vascular systems. Although TiO2 NPs are known to induce oxidative stress and inflammation, the associated signaling pathways have not been well studied. The focus of this work, therefore, deals with examination of the cellular signaling pathways responsible for TiO2 NP-induced endothelial oxidative stress and inflammation. In this study, primary vascular endothelial cells were treated with TiO2 NPs for 2–16h at concentrations of 0–50μg/mL. TiO2 NP exposure increased cellular oxidative stress and DNA binding of NF-κB. Further, phosphorylation of Akt, ERK, JNK and p38 was increased in cells exposed to TiO2 NPs. TiO2 NPs also significantly increased induction of mRNA and protein levels of vascular cell adhesion molecule-1 (VCAM-1) and mRNA levels of monocyte chemoattractant protein-1 (MCP-1). Pretreatment with inhibitors for NF-κB (pyrrolidine dithiocarbamate), oxidative stress (epigallocatechin gallate and apocynin), Akt (LY294002), ERK (PD98059), JNK (SP600125) and p38 (SB203580) significantly attenuated TiO2 NP-induced MCP-1 and VCAM-1 gene expression. These data indicate that TiO2 NPs can induce endothelial inflammatory responses via redox-sensitive cellular signaling pathways.

EGCG protects endothelial cells against PCB 126-induced inflammation through inhibition of AhR and induction of Nrf2-regulated genes

Tea flavonoids such as epigallocatechin gallate (EGCG) protect against vascular diseases such as atherosclerosis via their antioxidant and anti-inflammatory functions. Persistent and widespread environmental pollutants, including polychlorinated biphenyls (PCB), can induce oxidative stress and inflammation in vascular endothelial cells. Even though PCBs are no longer produced, they are still detected in human blood and tissues and thus considered a risk for vascular dysfunction. We hypothesized that EGCG can protect endothelial cells against PCB-induced cell damage via its antioxidant and anti-inflammatory properties. To test this hypothesis, primary vascular endothelial cells were pretreated with EGCG, followed by exposure to the coplanar PCB 126. Exposure to PCB 126 significantly increased cytochrome P450 1A1 (Cyp1A1) mRNA and protein expression and superoxide production, events which were significantly attenuated following pretreatment with EGCG. Similarly, EGCG also reduced DNA binding of NF-κB and downstream expression of inflammatory markers such as monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1) after PCB exposure. Furthermore, EGCG decreased endogenous or base-line levels of Cyp1A1, MCP-1 and VCAM-1 in endothelial cells. Most of all, treatment of EGCG upregulated expression of NF-E2-related factor 2 (Nrf2)-controlled antioxidant genes, including glutathione S transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), in a dose-dependent manner. In contrast, silencing of Nrf2 increased Cyp1A1, MCP-1 and VCAM-1 and decreased GST and NQO1 expression, respectively. These data suggest that EGCG can inhibit AhR regulated genes and induce Nrf2-regulated antioxidant enzymes, thus providing protection against PCB-induced inflammatory responses in endothelial cells.

Interaction of Protein Kinase C‐related Kinase (PRK) 1 with the TPα and TPβ isoforms of the human Thromboxane A2 receptor: Implications for Prostate Cancer

In humans, thromboxane (TX)A2 signals through the TPα and TPβ isoforms of the TXA2 receptor (TP). Here, the RhoA effector protein kinase C‐related kinase (PRK) 1 was identified as an interactant of both TPα and TPβ involving common and unique sequences within their respective carboxyl‐termini and the kinase domain of PRK1. While the interaction with PRK1 is constitutive, agonist‐activation of TPα/TPβ did not regulate the complex but enhanced PRK1 activation leading to phosphorylation of histone H1 in vitro. Altered PRK1/TP expression & signaling are increasingly implicated in certain neoplasms, particularly in androgen‐associated prostate carcinomas. Agonist‐activation of TPα/TPβ led to phosphorylation of histone H3 at Thr11 (H3Thr11), a recognized marker of androgen induced‐chromatin remodeling, in the prostate LNCaP and PC‐3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen‐responsive LNCaP but not in non‐responsive PC‐3 cells. Disruption of PRK1 impaired TPα/TPβ‐mediated H3Thr11 phosphorylation in, and cell migration of, both cell types. Given the role of TXA2 as a potent mediator of RhoA signaling, the identification of PRK1 as a bone fide interactant of TPα/TPβ has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated. Funded by SFI and HRB.